PMID:20667834

Citation	Gersting, SW, Staudigl, M, Truger, MS, Messing, DD, Danecka, MK, Sommerhoff, CP, Kemter, KF and Muntau, AC (2010) Activation of phenylalanine hydroxylase induces positive cooperativity toward the natural cofactor. J. Biol. Chem. 285:30686-97
Abstract	Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.
Links	PubMed PMC2945563 Online version:10.1074/jbc.M110.124016
Keywords	Allosteric Regulation/genetics; Biopterin/analogs & derivatives; Biopterin/chemistry; Biopterin/metabolism; Biopterin/therapeutic use; Coenzymes/chemistry; Coenzymes/metabolism; Coenzymes/therapeutic use; Enzyme Activation/genetics; Fluorescence; Humans; Kinetics; Mutation, Missense; Phenylalanine/chemistry; Phenylalanine/genetics; Phenylalanine/metabolism; Phenylalanine Hydroxylase/chemistry; Phenylalanine Hydroxylase/genetics; Phenylalanine Hydroxylase/metabolism; Phenylketonurias/drug therapy; Phenylketonurias/enzymology; Phenylketonurias/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:PH4H		GO:0004505: phenylalanine 4-monooxygenase activity	ECO:0000314:		F		Figure 1- Shows measurements of PAH activity based on different levels of substrate and cofactors.	complete CACAO 4047
HUMAN:PH4H	enables	GO:0004505: phenylalanine 4-monooxygenase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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